A series of isotope dilution and incorporation experiments has been completed in growing cultures of Neurospora crassa which delineate latter steps of the carnitine biosynthetic pathway as follows: epsilon-N-trimethyllysine yields beta-hydroxytrimethyllysine yields gamma-N-trimethylaminobutyraldehyde yields gamma-butyrobetaine yields carnitine. Unpublished work in our laboratory indicates the presence of lipid-bound lysine metabolites both in the rat and Neurospora. Work will focus on elucidating the chemical nature of such lipid metabolites and in determining the relationship, if any, of such compounds to carnitine synthesis and/or function. Lipid bound lysine will be isolated in sufficient quantities to permit studies of its methylation by S-adenosylmethionine in rat liver systems, as the biosynthesis of trimethyllysine relevant to carnitine synthesis in mammalian systems in not clear. Data are accumulating in our laboratory that many factors including age, sex, nutrition, and disease affect tissue levels of carnitine in man and animals. Such findings may concern aberrations in carnitine synthesis in the liver and/or its transport and uptake by the tissues. Delineation of the enzymology of the carnitine pathway will provide the methodology to address the question of impaired carnitine synthesis. Preliminary experiments in our laboratory indicate the presence of carnitine binding protein(s) in rat liver, plasma, cardiac muscle, skeletal muscle and the epididymis. Such proteins will be isolated and characterized and provide a means to consider aberrations of carnitine transport in the instances cited above.